The chronic consumption of alcoholic beverages is the major cause of serious liver disease. Ethanol is an extremely potent hepatoxin and can lead to cirrhosis of the liver upon prolonged exposure. In fact 20% of chronic alcoholics will eventually experience cirrhosis. The process of cirrhosis of the liver involves a series of steps beginning with fatty infiltration which leads to necrosis or cell death, then fibrosis which in turn leads to cirrhosis. Despite considerable research in this area, the underlying pathogenic mechanisms of ethanol induced liver injury, including the underlying biochemical reactions, remain obscure.
In recent years extensive evidence has come forward supporting a role of acetaldehyde in the detrimental actions of ethanol in liver as well as other organs. Numerous studies have shown that acetaldehyde can react with proteins in vitro under physiological conditions to form both stable and unstable adducts. Because of this chemical reactivity, the covalent binding of acetaldehyde to hepatic proteins has been proposed as a key event leading to alcohol liver injury. Many groups have demonstrated by immunoassays, using antibodies directed against acetaldehyde-modified protein, the presence of acetaldehyde adducts in the livers of rats, guinea pigs, and humans chronically consuming ethanol.
Studies involving the chemistry of acetaldehyde protein adduct formation have shown that acetaldehyde forms both unstable and stable adducts and that the xcex5-amino group of lysine participates in binding and, further, that unstable adducts serve as intermediates in stable adduct formation. It has also been found that proteins contain lysine residues with varying reactivities towards acetaldehyde adduct formation and that certain proteins (such as xcex1-tubublin) may be selective targets for adduct formation by virtue of containing a specially reactive xe2x80x9ckeyxe2x80x9d lysine residue. Further information about acetaldehyde adducts is present in Tuma, D. J. xe2x80x9cThe Role of Acetaldehyde Adducts in Liver Injuryxe2x80x9d, Hall P. Editors, Alcoholic Liver Disease Pathology and Pathogenesis, Ed. 2, London: Edward Arnold, 1995, 89-99 incorporated herein by reference.
The nature and/or chemical structures of acetaldehyde adducts that form in vivo have not been characterized and conflicting results in the literature concerning the nature, subcellular distribution, and identity of these adducts have been reported. See Tuma, D. J. xe2x80x9cThe Role of Acetaldehyde Adducts in Liver Injuryxe2x80x9d, Hall P. Editors, Alcoholic Liver Disease Pathology and Pathogenesis, Ed. 2, London: Edward Arnold, 1995, 89-99 previously incorporated herein by reference.
Another reactive aldehyde involved in alcohol liver injury is malondialdehyde (MDA). Malondialdehyde is formed by the peroxidation of polyunsaturated fatty acids and from the oxidative degradation of deoxyribose by a hydroxy radical. MDA is also produced in mammalian tissues as a side product of prostaglandin and thromboxane biosynthesis.
Several studies have suggested that chronic ethanol consumption induces hepatic lipid peroxidation which in turn, generates malondialdehyde. MDA is toxic, mutagenic, and inactivates enzymes due to modification of lysine residues. MDA protein adducts have been detected in the liver following administration of agents that promote lipid peroxidation such as carbon tetrachloride, iron overload, and more recently chronic ethanol feeding. It has been shown to form an adduct with a lysine residue (xcex5-amino group) of proteins and that MDA reacts with a primary amine to give a 1:1 Schiff base. For further information about MDA protein adducts see Houglum et al., J. Clin. Invest., 86: 1991 (1990) incorporated herein by reference.
Similar concentrations of acetaldehyde and MDA can co-exist in the liver during ethanol metabolism, as such both acetaldehyde and MDA adducts have been detected in livers of ethanol fed animals. Ohya demonstrates that malondialdehyde, in the presence of alkanals formed an adduct with the primary amine n-hexylamine. Ohya Parm. Bull. 16(2)137-141, February 1993. Due to the high levels of acetaldehyde and the production of malondialdehyde following alcohol ingestion applicants have discovered that the two aldehyde compounds chemically combine in a synergistic manner creating a new hybrid adduct formed with hepatic proteins. The hybrid adduct has been termed herein as malondialdehyde-acetaldehyde adduct (MAA). Although both aldehydes alone are capable of adduct formation with proteins, the influence of the presence of both acetaldehyde and MDA on adduct formation with complex proteins and their role in liver damage has not been addressed.
It is an object of the present invention to provide a novel protein adduct which occurs when malondialdehyde and acetaldehyde are combined and to delineate the role of both in adduct formation.
It is yet another object of the invention to provide a marker for alcohol liver damage which can be used to indicate the presence of liver disease, or other diseases with increased lipid peroxidation, lipids and/or or acetaldehyde which can include but is not limited to atherosclerosis or fat content for animals.
It is yet another object of the present invention to provide polyclonal and monoclonal antibodies which detect the presence of the novel acetaldehyde malondialdehyde protein adduct of the invention which can be used to assay for its presence.
It is yet another object of the present invention to define these novel acetaldehyde malondialdehyde protein adducts by chemical formulation.
It is yet another object of the invention to provide methods of use for the novel acetaldehyde malondialdehyde protein adduct in immunoassays such as FITC, FACS, and Western Blotting.
This invention discloses a novel reactant product of acetaldehydeand malondialdehyde (MDA) which interact together forming a novel compound which is highly reactive and is also adducted to antigens including complex proteins, lipids, carbohydrates or DNA simply by incubation of the two aldehydes with the antigen. The combination of MDA and acetaldehyde in the presence of various antigens causes a formation of a new distinct product comprising a hybrid adduct of MDA and acetaldehyde which has been designated malondialdehyde, acetaldehyde-adduct (MAA). These hybrid adducts are novel and the general chemical formula has been characterized. This is in stark contrast to most adducts which have not been so delineated, and provides opportunities for independent synthesis of the adduct itself and for creation of new antigen adduct combinations, as well as alternative assay methods.
According to the invention, the combination of MDA and AA in the presence of a protein or peptide creates two hybrid adducts which have been characterized at the molecular level having the following formulas. The hybrid adducts of the invention can include alterations at various functional groups which would not be expected to change the overall reactivity of the product. The formula for these hybrid adducts is listed below. 
Wherein X is an antigen which contains a reactive amino residue, and can be a peptide, protein, DNA molecule, carbohydrate or lipid. Compound I is associated with the presence of ALD and may be a better marker than compound II (MAA) except that it is less stable. 
Wherein R is a lower alkyl from C1 to about C6, H, or benzyl group and X is an antigen which contains a reactive amino residue, and can be a peptide, protein, DNA molecule, carbohydrate or lipid. These hybrid adducts are formed under standard conditions, by simple incubation.
In addition to the above-identified novel hybrid adducts, the adducts of the invention have several important immunological properties which can be exploited for further chemical and immunological assay procedures. Monoclonal and polyclonal antibodies have been produced which recognize these adducts and can be used to identify them as markers of alcohol liver disease or other diseases associated with increased lipid peroxidation, lipids, and/or acetaldehyde such as atherosclerosis and fat content for domestic animals.
MAA is highly reactive and will bind antigens preferentially to either acetaldehyde or malondialdehyde alone. Thus, MAA is a more sensitive indicator of the presence of concomitant liver disease. MAA also causes adducted proteins to become highly immunogenic. When used as an antigen, it produces high antibody titre and acts as a specific immune enhancing agent obviating the need for adjuvant. Without wishing to be bound by any theory it is postulated that MAA aids in antigen presentation of adducted proteins stimulating an immune response.
The MAA reaction product is highly fluorescent and the fluorescence is observed only when both aldehydes are co-incubated with the proteins. The fluorescence is large, has an excitation frequency of 398 nanometers and an absorbance of 460 nanometers. The fluorescence can be used as a label, allowing for detection of fluorescence at a picomolar range, enabling direct detection of antigen antibody complexes.